Genetic Diversity of SCN5a Gene

Hindawi Publishing Corporation
BioMed Research International

Volume 2014, Article ID 462609, 13 pages
http://dx.doi.org/10.1155/2014/462609
Research Article
Genetic Diversity of SCN5A Gene and Its Possible Association
with the Concealed Form of Brugada Syndrome Development in
Polish Group of Patients
Beata UziwbBo-gyczkowska,1 Grzegorz Gielerak,1 PaweB Siedlecki,2 and Beata Pajdk3,4
1
Department of Cardiology and Internal Diseases, Military Institute of Medicine, Szaserow Street 128, 04-141 Warsaw 44, Poland
Department of Bioinformatics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
3
BioVectis/Kucharczyk TE, Pawinskiego 5A, 02-106 Warsaw, Poland
4
Electron Microscopy Platform, Mossakowski Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw, Poland
2
Correspondence should be addressed to Beata Uzibo-Zyczkowska;

buzieblo-zyczkowska@wim.mil.pl
Received 29 April 2014; Revised 1 August 2014; Accepted 10 August 2014; Published 20 October 2014
Academic Editor: Diego Franco
Copyright 2014 Beata Uzibo-Zyczkowska

et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Brugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The paper presents
the discovery of new genetic variants of SCN5A gene which might be associated with the development of a concealed form of
Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome.
Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test results were
subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples
obtained from the family members with Brugada Syndrome, despite the fact that they had negative ajmaline challenge test results.
Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point
(SNP) variants located at exons, and 12 new single nucleotide point variants located at introns. Among new SNPs localized in
SCN5A gene exons three SNPs affected the protein sequence.
1. Introduction
About 510% of sudden cardiac death (SCD) cases are caused
mainly by electrical heart diseases [1]. In the recent years, a
special attention has been paid to one of them, the Brugada
Syndrome (BS). The diagnosis of BS is based on ECG criteria
as well as on the clinical picture. Typical BS ECG changes
occur as a result of ion function disorders in the heart,
which are caused by genetic mutations and lead to improper
course mainly of repolarization processes in cardiomyocytes.
Electrocardiographic features of the syndrome are dynamic
and ECG curve is periodically normal; typical BS characteristics disappear, which makes it difficult to diagnose
BS. Concealed form of BS causes underestimation of this
diseases occurrence frequency and a great number of people
remain undiagnosed. Specific pharmacological provocation
tests with class I medicine are critical in revealing concealed

ECG features of BS.
1.1. Genetic Background of the BS. The first gene to be linked
to BS is the SCN5A, the gene that encodes the -subunit of
the cardiac sodium channel gene [2]. Almost 400 mutations
at the SCN5A gene have been identified at the syndrome
patients since 2001 [3, 4]. Numerous detected mutations have
been studied at the functional level [5]. The mutations at the
SCN5A gene occur in approximately 18% to 30% of Brugada
Syndrome cases. A higher incidence of SCN5A mutations has
been reported in familial rather than in sporadic cases [6].
Other gene loci on chromosome 3, which is close to
but distinct from SCN5A, have recently been linked to the
syndrome (3p22p24) [6] and GPD-1L [7]. Those mutations
resulted in the loss of function of the cardiac sodium channel.

Other genes associated with BS were reported in the last few
years and shown to encode the 1 and subunits of the L-type
cardiac calcium channel [8].
The SCN5A gene remains the main gene linked to BS. Of
note, negative SCN5A results generally do not rule out causal
gene mutations. Currently, knowledge of a specific mutation
may not provide guidance in formulating a diagnosis or
determining a prognosis. Mutation screening of the SCN5A
gene in patients with BS may only support a clinical overt or
suspicious diagnosis.
In recent years, the genotyping of SCN5A gene was more
correlated to the prognostic value than to the diagnosis of
the BS itself. Some of the SCN5A mutations were related to a
worse clinical course [9] and others to a better [10] prognosis
of the BS patients.
2. Materials and Methods

2.1. Patient Populations. The study involved a group of 59
Polish patients (37 men) with suspected concealed BS based
on specific ECG and/or clinical criteria:
(i) complete and incomplete right bundle branch block
(RBBB) in ECG,
(ii) suspected but nondiagnostic ECG (types 2 and 3),
(iii) history of sudden cardiac arrest (SCA),
(iv) unexplained syncopes,
(v) sudden cardiac death (SCD) amongst family members under 45,
(vi) family history of BS.
The protocol of the study has been approved by the
Commission for Bioethics. Written informed consent was
obtained from all of the patients.
2.2. The Ajmaline Challenge Test. All patients were performed
with pharmacological provocation with intravenous ajmaline
administration dosed 1 mg/kg body weight for 5 min, in safe
conditions during 12-lead 24-hour Holter ECG monitoring.
In patients with positive test results, molecular tests of the
SCN5A gene were performed.
Molecular tests were performed in the family members of
patients with BS, even if the pharmacological provocation test
was negative in these individuals.
Occurrence of type 1 electrocardiographic patterns (coveshaped ST elevation in right precordial leads with J wave or
ST elevation of 2 mm (mV) at its peak followed by a negative
T wave with little or no isoelectric interval in more than one
right precordial lead, V1V3) or conversion of type 2 or 3
to the diagnostic type 1 pattern after ajmaline administration
was considered as a positive test result [3]. Occurrence of type
2 or 3 ST segment elevation was considered as negative test
result.
2.3. DNA Analysis. The genetic analysis was conducted in
collaboration with Kucharczyk TE/BioVectis Company (Warsaw, Poland). Genomic DNA were analysed in 7 patients
with positive result of ajmaline challenge test (one patient

with positive result of ajmaline challenge test did not agree
to be genotyped) and in 1 family member of patients with
negative result of ajmaline challenge test. Genomic DNA
were extracted from peripheral blood leucocytes (100 L of
frozen blood was used). Isolation was performed according
to the manufacturers protocol (A&A Biotechnology, Poland).
Regions most likely to contain genetic mutations at 28 exons
of the SCN5A gene were covered by 41 PCR amplicons,
covering 28 exons and partial intron sequences, as previously
described [11]. Several pairs of primers were synthesized
to amplify with PCR reaction exons 12, 17, and 28 due to
their large sizes, named as 12a, 12b, 17a, and so forth. PCR
primers were designed to cover the full coding sequence
(exons), as well as partial fragments of flanking noncoding
fragments (introns). The PCR products were separated on
agarose gel to examine their specificity and to normalise the
DNA concentration. Next, 328 PCR products were screened
by multitemperature single-strand conformation polymorphism (MSSCP) [12] method for the presence of a singlepoint mutation or a polymorphism. The MSSCP conditions
were individually optimized for each PCR product. MSSCP
was performed on 7 to 10% T polyacrylamide gel, 3.3% C
at 0.75x TBE buffer. For some regions, glycerol was added
to polyacrylamide gel up to 5% w/v concentration. MSSCP
analysis was performed using DNA Pointer System in 0.5x
TBE buffer. Temperature profile of electrophoresis was 35
155 C. Electrophoresis was performed with 40 W of electrical power. Before applying samples onto the gel, 10 min
of preelectrophoresis (40 W at 35 C) was performed. At the
beginning, samples were maintained for 10 min at 100 V for
concentration. Subsequently, MSSCP separation was made.
The PCR products that have altered MSSCP mobility were
followed by Sanger method. 20 ng DNA of PCR products
were used as a matrix for sequencing reaction. Both strands
were sequenced at PCR products that revealed a genetic
alternation. Genetic alterations were identified using the
BLAST (Basic Local Alignment Search Tool) program and its
BLASTN version as well as UCSC (University of California
Santa Cruz) Genome Bioinformatics and NCBI (National
Center for Biotechnology Information) databases of singlenucleotide polymorphisms (SNPs).
2.4. Functional Analysis of SCN5A Variants. An in silico analysis was performed to evaluate the putative functional impact
of the three identified variations (S321Y, S519F, and K974D).
We used the Polymorphism phenotyping-2 (PolyPhen-2)
server [13], which integrates sequence-based and structurebased features to predict amino acid substitution effects
using a nave Bayes classifier. An amino acid change was
classified as probably damaging if its probability score
was greater than 0.85 or as possibly damaging if the
score was between 0.85 and 0.55. To assess the influence
of putative unstructured regions, we used DISOPRED3 [14]
software along with DOMPRED [15] to predict possible
domain boundaries and disordered binding regions. Finally,
we used Phyre [16] for structural feature predictions, mainly
Table 1: Distribution of the examined population depending on

inclusion criteria.
Inclusion criteria
RBBB in ECG (complete

and incomplete)
Number of included
patients
35 patients (59.32%)
(i) RBBB complete8
patients (13.6%)
(ii) RBBB incomplete27
patients (45.76%)
History of SCA
7 patients (11.8%)
Unexplained syncopes
SCD amongst family
members under 45
Family history of Brugada
Syndrome
31 patients (52.5%)
Suspected but
nondiagnostic ECG (types
2 and 3)
5 patients (8.5%)
4 patients (6.8%)
16 patients (27.11%)
(i) Type 24 patients
(6.78%)
(ii) Type 312 patients
(20.33%)
transmembrane regions and secondary structure using three

different algorithms.
3. Results
3.1. Patient Demographics. Study inclusion criteria were met
by 59 patients (22 women and 37 men) (Table 1). Average age
of the group was 31.612.2 years, from 16 to 62 years. Average
age for women was 29.68 10.9 years while, for men, it was
32.8 12.9 years. The majority of patients (72.8%) were under
40.
Echocardiography in all the included patients revealed no
significant organic heart disease.
3.2. Clinical Characteristics of the Group with Positive Result of
Pharmacological Provocation Test. Pharmacological provocation test was carried out on the whole study group.
No significant undesirable effects were observed. None of
the patients met the criteria of discontinuation prior the
scheduled conclusion of the study.
Positive test resulttype 1 ST segment elevation
(Figure 1(a)), which was considered as diagnostic of BS, was
obtained in 7 individuals (11.86%). The other 52 patients
(88.14%) had negative provocation test results (Figure 1(b)).
The group of 7 patients with type 1 ST segment elevation
diagnostic of BS following ajmaline administration consisted
of 6 men (85.7%) and 1 woman (14.3%). Average age of this
group was 36.5 15.2, from 16 to 52 years. The group of
patients with negative test results included 31 men (59.6%)
and 21 women (40.4%). Average age of this group was
30.9 11.7, from 18 to 62 years. No statistically significant
correlation between gender, age, or body mass and ajmaline
test was observed.
As regards the group of 7 individuals with positive
provocation test results, 2 patients had history of SCA (men),
among whom, in 1 person, the diagnosed SCA mechanism
was ventricular fibrillation. The SCA mechanism in the

second individual remains unknown. Both patients were
implanted a cardioverter-defibrillator. Within the group of
the other 5 patients, initially considered as asymptomatic, 16
months following the provocation test, syncopes occurred in
1 person (woman), which was an indication of implanting
a cardioverter-defibrillator. The other 4 individuals have
remained asymptomatic during the observation period lasting from 39 to 60 months.
3.3. Results of MSSCP Analysis and DNA Sequencing of
the SCN5A Sodium Gene. Genetic examinations of SCN5A
gene showed 6 known polymorphisms: rs6599230 (A>G,
A29A), rs41312393 (A>G, intron), rs1805126 (T>C, A1818G),
rs7429945 (A>G, exon, nontraslanted region), rs41315485
(T>C, exon, nontraslanted region), and rs7430407 (A>G,
E1061E). Three of them were noted at regions of coding
proteins, two at noncoding regions and one at intron. Numerous new genetic variants were detected: at nontraslanted
regions (8 SNPs), at introns (12 SNPs), and in the protein
coding regions (5 SNPs)2 DNA sequence variants caused
no change in the coded amino acid, whereas 3 altered the
coded amino acid.
An example of MSSCP analysis of 2 amplicones representing exons number 2 and 7 of the SCN5A gene for 8
particular patients is presented in Figure 2. On the other
hand, Figure 2 shows an example of patients derievedamplicone sequence analysis, which was compared with
reference sequences (Figure 3). All detected polymorphisms
were further analyzed in context of their localization and its
impact on aa SCN5A protein sequence.
3.4. Known Polymorphisms. The rs6599230 polymorphism at
exon 2 of the SCN5A gene was found in 2 patients related to
each other. It involved an alteration of nucleotides in 38614716
position (A>G) of reference sequence; however, detected
variant did not alter the aa in protein sequence (A29A), thus
having no impact on protein function. The patients with
this variant were a man (father) and a woman (daughter),
both asymptomatic. The pharmacological provocation test
was positive in the man and negative in the woman.
On the other hand, in exon 17b of the SCN5A gene, a
known rs7430407 polymorphism was identified in 1 person. It
involved a nucleotide alteration in 38562471 position (A>G)
of reference sequence. The patient with this variant was a man
with asymptomatic BS diagnosed based on pharmacological
provocation. This genetic alteration caused no amino acid
changes in protein sequence (E1061E).
Genotyping exon 24 of the SCN5A gene revealed a known
rs41312393 polymorphism in 3 individuals. It involved an
alteration in nucleotides in 38538672 position (A>G) of
reference sequence and was located at intron. The 3 persons
were asymptomatic2 men with positive pharmacological
provocation test and a woman related to one of the men
(daughter) with a negative result.
At exon 28c of the SCN5A gene a known 1805126 polymorphism was identified in 4 patients. This genetic change
involved a nucleotide alteration in 38532410 position (T>C)
(a)
(b)
Figure 1: (a) 12-lead ECG from a patient with positive test result (before and after test). The configuration of the ST segment elevation in
leads V1 to V3 is a coved type. (b) 12-lead ECG from a patient with negative test result (before and after test).
of reference sequence and caused no change in the amino

acid sequence in the coded protein (D1818D). Clinically,
there were 2 asymptomatic individuals related to each other
(father and son); one had negative result of pharmacological
provocation (father) whereas the second patient had type 1
change in ST segment typical of BS. The other 2 patients were
not related; one was a man with symptomatic BS and with
history of SCA while the other was a man with asymptomatic
BS.
Further, analysis of exon 28f of the SCN5A gene revealed
the presence of known rs7429945 polymorphism, which was
detected in 7 patients. It involved a nucleotide alteration in
38531693 position (A>G) of reference sequence. This genetic
change occurred in the nontraslanted part of the exon in 6365
mRNA position. The described genetic change was present in
almost every patient. Its presence was not observed only in a
man with symptomatic BS and history of SCA.
Another known polymorphism is rs41315485 identified
in 6 patients at exon 28k of the SCN5A gene. It involved
an alteration in nucleotides in 38530279 position (T>C) of
reference sequence, in 7779 mRNA position, and was located
in the nontraslanted region. The polymorphism was not
observed only in 2 individuals from the analyzed group. They
were men (brothers)one with symptomatic BS and the
other with asymptomatic BS.
3.5. New Genetic Variants in Nontraslanted Regions at Exons.

At exon 1 of the SCN5A gene, a new polymorphism that
involved an alteration in nucleotides in 38631119 position
(G>A) of reference sequence in 49 mRNA position was
observed. The change was connected with the region transcribed on mRNA but is not translated as a protein. The
person with this genetic variant was a man with diagnosed
symptomatic BS (with history of SCA). The polymorphism
was not observed in other patients.
Another new DNA sequence change was observed in 2
patients who were related to each other. It was connected
with the change in nucleotides in 38614815 position (G>C)
of reference sequence found at exon 2. The genetic variant
Exon 2
4 5 6
Exon 7
4 5 6
Figure 2: MSSCP separation of exon 2 and exon 7 PCR products.

Note that samples number 5 and number 7 at exon 2 and sample
number 1 at exon 7 have distinct electrophoretic profiles suggesting
the presence of minor genetic variants.
was present in 182 mRNA position and was related to a nontraslanted mRNA part. The patients with this polymorphism
(a woman and a man) were asymptomatic; pharmacological
provocation test was positive in the man and negative in the
woman.
A new DNA sequence change was also observed at exon
28g of the SCN5A gene in 5 patients. It involved an alteration
in nucleotides in 38531355 position (G>A) of reference
sequence in 6703 mRNA position. The genetic alteration was
present in a nontraslanted part of the exon. The 5 individuals
included 2 men with symptomatic BS and history of SCA.
Moreover, the group included the brother and the father of
the patient with history of SCA, one with a negative and the
other with a positive result of pharmacological provocation
test. The last person with this polymorphism was a man with
asymptomatic BS.
Genotyping exon 28i of the SCN5A gene revealed 4
new polymorphisms localized in a nontraslanted part of the
exon. These changes were found in all the examined persons,
among whom 4 patients had all the 4 genetic variants and 3
patients had two new sequence changes: in 38530974 (C>T)
and 38531102 (C>T) position of reference sequence, while
polymorphism in 38530974 position (C>T) was observed in
all the patients.
Furthermore, analysis of exon 28l of the SCN5A gene
showed a new sequence variant in 38529996 position (C>G)
of reference sequence in 8062 mRNA position. The change
Exon 2 ref.
Exon 2 samp.
Consensus
5
1
11
21
31
41
51
GGTCTGCCCACCCTGCTCTCTGTCCCTGGGCATAGAATCAGGCCCATTGTCTGTGTCTTC
GGTCTGCCCACCCTGCTCTCTGTCCCTGGGCATAGAATCAGGCCCATTGTCTGTGTCTTC
ggtctgcccaccctgctctctgtccctgggcatagaatcaggcccattgtctgtgtcttc
61
71
81
91
101
111
GAGCTTCCCCACAGGCAACGTGAGGAGAGCCTGTGCCCAGAAGCAGGATGAGAAGATGGC
GAGCTTCCCCACAGGCAACGTGAGGAGAGCCTGTGCCCAGAACCAGGATGAGAAGATGGC
Exon 2 ref.
Exon 2 samp. 1
Consensus
cagcttccccacaggcaacgtgaggagagcctgtgcccagaa caggatgagaagatggc
Exon 2 ref.
Exon 2 samp. 1
Consensus
121
131
141
151
161
171
AAACTTCCTATTACCTCGGGGCACCAGCAGCTTCCGCAGGTTCACACGGGAGTCCCTGGC
AAACTTCCTATTACCTCGGGGCACCAGCAGCTTCCGCAGGTTCACACGGGAGTCCCTGGC
aaacttcctattacctcggggcaccagcagcttccgcaggttcacacgggagtccctggc
Exon 2 ref.
Exon 2 samp. 1
Consensus
Exon 2 ref.
Exon 2 samp. 1
Consensus
Exon 2 ref.
Exon 2 samp. 1
Consensus
Exon 2 ref.
Exon 2 samp. 1
Consensus
Exon 2 ref.
Exon 2 samp. 1
Consensus
181
191
201
211
221
231
AGCCATCGAGAAGCGCATGGCAGAGAAGCAAGCCCGCGGCTCAACCACCTTGCAGGAGAG
AGCCATCGAGAAGCGCATGGCGGAGAAGCAAGCCCGCGGCTCAACCACCTTGCAGGAGAG
agccatcgagaagcgcatggc gagaagcaagcccgcggctcaaccaccttgcaggagag
241
251
261
271
281
291
CCGAGAGGGGCTGCCCGAGGAGGAGGCTCCCCGGCCCCAGCTGGACCTGCAGGCCTCCAA
CCGAGAGGGGCTGCCCGAGGAGGAGGCTCCCCGGCCCCAGCTGGACCTGCAGGCCTCCAA
ccgagaggggctgcccgaggaggaggctccccggccccagctggacctgcaggcctccaa
311
321
331
341
351
301
AAAGCTGCCAGATCTCTATGGCAATCCACCCCAAGAGCTCATCGGAGAGCCCCTGGAGGA
AAAGCTGCCAGATCTCTATGGCAATCCACCCCAAGAGCTCATCGGAGAGCCCCTGGAGGA
aaagctgccagatctctatggcaatccaccccaagagctcatcggagagcccctggagga
361
371
381
391
401
411
CCTGGACCCCTTCTATAGCACCCAAAAGGTGACTACCACCCACCTCCAGCCCTGCCTACC
CCTGGACCCCTTCTATAGCACCCAAAAGGTGACTACCACCCACCTCCAGCCCTGCCTACC
cctggaccccttctatagcacccaaaaggtgactaccacccacctccagccctgcctacc
421
431
CTTCTGTGCAACTCCC
CTTCTGTGCAACTCCC
cctctgtgcaactccc
441
451
461
471
Figure 3: Sequence alignment of SCN5 exon 2 reference (WT) and MT amplicon detected in sample 5 (pos. 38614716 heterozygote A>G,
exon, amino acid pos. 29, polymorphism rs6599230; pos. 38614815 heterozygote G>C, exon, pos. 182 in mRNA, nontraslanted region). White
color shows changed nucleotides.
was found only in 1 person. Clinically, the person with this

polymorphism was a man with asymptomatic BS.
3.6. New Polymorphisms in Protein Coding Regions That Cause
No Alteration in the Coded Amino Acid. As regards the group
of 8 examined patients, 2 unknown genetic variants were
observed in one patient at exon 28c of the SCN5A gene in
protein coding regions; they caused no change in the coded
amino acid; thus, we should consider them a polymorphic
change. The two novel polymorphic variants were detected
in positions 38532614 (C>T, F1750F) and 38532617 (C>T,
L1749L). In both cases, the changed nucleotide is in the 3rd
coded position, which may influence the fact that it causes no
alteration in the amino acid sequence. These genetic variants
were observed in a patient with diagnosed BS and history of
SCA who required implantation of cardioverter-defibrillator.
3.7. New Sequence Changes in the Protein Coding Regions That
Alter the Coded Amino Acid. During genetic analysis of the
SCN5A gene, presence of 3 unknown genetic variants that

altered the coded amino acid was noticed in 5 patients.
The first variant contained change in nucleotides in
38589682 position (C>A) of reference sequence and was
observed in 1 patient with negative result of pharmacological
provocation. This genetic change was observed in exon 8 of
the SCN5A gene. It altered serine amino acids into tyrosine
in 321 position of the coded protein (S321Y). This variant was
found in none of the other patients.
Another genetic variant detected in one patient involved
a change in nucleotides in 38585541 position (C>T) of
reference sequence, which altered serine amino acids into
phenylalanine in 519 protein position (S519F). New variant
was found at exon 12 of the analyzed gene. The change was
observed in a patient with asymptomatic BS and its presence
was confirmed neither in 2 family members of the patient nor
in the other examined patients.
The last new sequence variant, which, according to the
UCSC Genomi Bioinformatic database, is found in the

DNA SNP
Detected aa
alterations (WT/MT)
SCN5A protein
ex 8,
C>A
ex 12,
C>T
ex 17,
G>T
S/Y
S/F
K/D
C
N
321
aa number
519
974
2015
Figure 4: Schematic illustration of Nav1.5, showing the location of the novel putative amino acid changes.
N-terminal
Extracellular
Cytoplasmic
133
180
193
243
247
374
389
737
751
798
808
861
915
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
150
160
210
220
273
359
416
718
770
781
833
837
942 Membrane
C-terminal
(a)
This mutation is predicted to be possibly damaging with a score of 0.667 (sensitivity: 0.86; specificity: 0.91)
S321Y
HumDiv
0.00
0.20
0.40
0.60
0.80
1.00
HumVar
0.00
0.20
0.40
0.60
0.80
0.60
0.80
1.00
This mutation is predicted to be probably damaging with a score of 0.992 (sensitivity: 0.49; specificity: 0.95)
HumVar
0.00
0.20
0.40
0.60
0.80
1.00
K974D
0.00
HumDiv
0.20
0.40
0.60
0.80
1.00
HumVar
0.00
0.20
0.40
0.60
0.80
1.00
(b)
Confidence score
0.40
Na trans assoc
9531215
0.8
HumDiv
S519F
0.20
DUF3451
461668
1.00
This mutation is predicted to be probably damaging with a score of 0.997 (sensitivity: 0.41; specificity: 0.98)
0.00
Ion trans
159412
0.6
0.4
0.2
200
400
600
800
1000
Amino acid position
1200
1400
(c)
Figure 5: (a) Prediction of transmembrane, extracellular in cytoplasmic regions in SCN5a protein. Location of the three new variants is
depicted as orange dots. (b) Prediction of functional effects of nonsynonymous mutations done in PolyPhen2 software. All three variants
have two scores from HumDiv (genomic oriented) and HumVar (diagnostic oriented). (c) Prediction of intrinsically disordered regions in
SCN5a (11400AA) done by DISOPRED3. Domain organisation shown with respect to disordered regions. Orange color marks putative
binding sites. High confidence score indicated better chance of unstructured fragment.
protein coding region, was observed in 4 patients. The

polymorphism was observed at exon 17 of the SCN5A gene
and involved a change in amino acids in 38562732 position
(G>T) of reference sequence. This genetic change altered
lysine amino acids into aspartic acid in 974 position of
the coded protein (K974D). BS was diagnosed in 2 of the

persons while the other 2 individuals were family members of patients with negative results of pharmacological
provocation. Schematic representation of detected changes is
illustrated in Figure 4. To evaluate the possible influence of

new missense mutations on channel function, bioinformatics
analysis has been conducted.
3.8. In Silico Functional Analysis of SCN5A Variants. The
three variants identified are located at the cytoplasmic region
of the SCN5A-encoded protein (Figure 5(a)). Confirmed
disease associated genetic variants can be found in close
proximity to each one of the new variants, as well as sites of
amino acid modifications (e.g., arginine methylation site at
513 and 526 or a glycosylation site at 318). This would hint
that the observed variants are located in important regions for
protein function. To further explore their possible functional
impact, we employed a well-known bioinformatics algorithm
PolyPhen2. The tool indicated a high possibility of damage
caused by mutating K974D with prediction score close to
1 (the highest possible) (Figure 5(b)). For the two other
mutations, possible damage was also reported, but with lesser
probability. We sought to confirm these predictions with
more structural insights. We used three different software
tools to establish whether these variations would occur in
unstructured and putative domain regions or not. Indeed,
S519F is located in a large domain of unknown function
(DUF3451, PFAM: PF11933), which is also predicted as an
unstructured/disorderd region by all three bioinformatics
methods (Figure 5(c)). This would suggest a possible protein
binding interaction in this region, which could be hampered
by this variant (especially since serine contains a hydroxylic
polar group and phenylalanine is hydrophobic and aromatic).
On the other hand, S321Y is also located in transmembrane
ion channel family domain (Ion trans, PF) which is predicted
to be structured. The same goes with K974D, located just at
the beginning of the sodium ion transport-associated domain
(Na trans assoc, PF06512). Again, this is a structured region
but very close to the predicted unstructured binding region
(945956).
3.9. New Point Mutations Found at Introns. In the regions
of the SCN5A gene, which, according to the UCSC Genome
Bioinformatics database, are at introns, 12 new point mutations were found.
MSSCP analysis of exon 4 of the SCN5A gene detected
4 new mutations within the intron. The first was a mutation
in 38603806 position of reference sequence and involved
a type A insertion. The second mutation was an alteration
in nucleotides in 38603801 position (T>A) of reference
sequence. The two said genetic changes were confirmed in
5 patients, among whom 3 were asymptomatic and 2 were
symptomatic (1 with history of SCA and 1 with syncopes).
Another genetic change detected at this exon in the other 2
individuals was a change in nucleotides in 38604076 position
(G>T) of reference sequence. The first patient had positive
provocation result and history of SCA whereas the second
patient was asymptomatic and also had positive provocation
result. The last sequence change at this exon was found
only in one patient in 38604075 position (G>T) of reference
sequence. The patient was asymptomatic with negative result
of ajmaline test.
7
MSSCP analysis showed presence of 2 new genetic variants at exon 6 of the SCN5A gene within the intron in 7
patients out of 8. The first involved a change in nucleotides
in 38595390 position (C>G) of reference sequence. The
second mutation was a change in 38595384 position (C>G)
of reference sequence. These DNA variants were observed in
all the examined patients apart from one individual who was
asymptomatic and had negative result of pharmacological
provocation test. At exon 7 of the SCN5A gene, a DNA
sequence variant within the intron (pos. 38591480, C>G) was
identified in 4 patients out of 6 with positive ajmaline provocation test. Moreover, 3 patients from this group had symptomatic BS and had an implanted cardioverter-defibrillator
either due to history of SCA or due to unexplained syncopes.
Concurrently, this polymorphism was confirmed in neither
of the patients with negative results of provocation test. The
results of MSSCP genotyping of 41 amplicons representing
SCN5A gene are summarized in Table 2. Additionally, Table 3
contains the list of intronic alterations and exchanges in
noncoding regions along with short stretches of sequence
alignments (WT > MT).
4. Discussion
The major gene related to BS is the SCN5A gene. Despite
the great development in molecular studies, it is estimated
that mutations in the SCN5A gene cause only about 18
30% of BS cases [17]. These mutations are more common
in familial cases of the disease than in sporadic ones [18].
Negative results of genetic studies do not exclude causal
gene mutations. Neither diagnosis nor prognosis of BS can
be based on genetic test results. In the presented work, a
molecular analysis of the whole SCN5A gene was carried out
with respect to patients with positive provocation test (apart
from 1 person who failed to give their informed consent)
as well as their family members (1st degree of kinship) who
gave their informed consent. Due to both low predicted BS
incidence in the Polish population (lack of accurate data) and
a considerably low percentage of the known genetic changes
being the underlying cause of the disease (1830% as above),
the work was limited only to analyzing the occurrence of
the known mutations. The molecular study of the 28 exons
and short exon/intron fragments of SCN5 gene was carried
out including also the alterations in the sequence of the few
noncoding regions of the gene (introns). In this study, the new
genetic variants were found both at exons and at introns. It is
a commonly accepted fact that the effects of DNA sequence
change depend on their location in the gene. However, all too
often, it is assumed that only genetic alterations in the coding
sequences, that is, at exons, have an impact on the clinical
course of the disease. Recent studies and findings have shown
that intronic mutations may play a major role in the splicing
process, alter its course, lead to coding sequence abnormalities, and consequently influence the structure and function
of the encoded proteins. Numerous data reported in scientific
papers show that both intronic and exonic alterations may
result in an aberrant splicing process, leading to the formation
of abnormal proteins, which, in turn, affects the severity of
Asymptomatic
Negative pharmacological
provocation test
Father of the patient with
diagnosed BS
History of sudden cardiac

arrest
Positive pharmacological
provocation test
Diagnosed BS
History of sudden cardiac

arrest
provocation test
Diagnosed BS
Patient 1
Patient 2
Patient 3
Major clinical data
Genetic alterations in DNA sequences of SCN5 amplicons

(reference sequence ref|NT 022517.17|Hs3 22673)
Amplicon 4: pos. 38603806, insertion A, intron
pos. 38603801 homozygote A, intron
Amplicon 8: pos.38589643 insertion A, intron
pos. 38589682 C>A, amino acid pos. 321, S>Y
Amplicon. 17a: pos. 38562732 G>T, amino acid pos. 974, K>D
Amplicon 28c: pos. 385924060 T>C, exon, amino acid pos. 1818 D>D, rs1805126
Amplicon 28f: pos. 38531693 A>G, exon, mRNA position 6365, near 3 UTR, nontraslanted region, rs7429945
Amplicon 28g: pos. 38531355 heterozygote G>A, mRNA position 6703, near 3 UTR, nontraslanted region
Amplicon 28i: pos. 38530974 C>T, exon, mRNA position 7084, near 3 UTR, nontraslanted region
pos. 38531102 C>T, exon, mRNA position 6956, near 3 UTR, nontraslanted region
Amplicon 28k: pos. 38530279 T>C, exon, mRNA pos. 7779, 3 UTR, nontraslanted region, polymorphism rs41315485
pos. 38603801 homozygote T>A, intron
Amplicon 6: pos. 38595390 G>C, intron
pos. 38595384 homozygote G>C, intron
Amplicone 7: pos. 38591480 C>G, intron
Amplicon 28g: pos. 38531355 G>A, exon, mRNA position 6703, near 3 UTR, nontraslanted region
Amplicon 28i: pos. 38530974 G>A, exon, mRNA position 7084, near 3 UTR, nontraslanted region
Amplicon 1: pos. 38631119 G>A, mRNA pos. 49, nontraslanted region
Amplicon 4: pos. 38604076 G>T, intron
Amplicon 6: pos. 38595390 homozygote C>G, intron
pos. 38595384 homozygote C>G, intron
Amplicon 7: pos. 38591480 C>G, intron
Amplicon 17a: pos. 38562732 G>T, amino acid pos. 974, K>D
Amplicon 28c: pos. 38532617 C>T, exon, amino acid pos. 1749, I>I
pos. 38532614 C>T, exon, amino acid position 1750, F>F
pos. 38592406 T>C, exon, amino acid pos. 1818, D>D, rs1805126
Amplicon 28g: pos. 38531355 heterozygote G/A, mRNA position 6703, near 3 UTR, nontraslanted region
Table 2: The results of MSSCP genotyping of 41 amplicons representing SCN5A gene.
8
Asymptomatic
provocation test
Brother of the patient with
diagnosed BS
Asymptomatic
provocation test
Father of the patient with
diagnosed BS
provocation test
Diagnosed BS
Patient 4
Patient 5
Patient 6
Major clinical data
Table 2: Continued.
Amplicon 4: pos. 38604076 G>T, intron
Amplicon 12a: pos. 38585541 C>T exon, amino acid pos. 519, S>F
pos. 38585647 homozygote A>T, intron
Amplicon 22: pos. 38544050, heterozygote C/T, intron
Amplicon 24: pos. 38547178 heterozygote A/G, intron, polymorphism rs41312393
Amplicon 28c: pos. 38592406 T>C, exon, amino acid pos. 1818, D>D, rs1805126
Amplicone 2: pos. 38614716 A>G, exon, amino acid pos. 29, A>A, polymorphism rs6599230
pos. 38614815, G>C, exon, mRNA pos.182, nontraslanted region
Amplicone 4: pos. 38604075 homozygote G>T, intron
Amplicon 17b: pos. 38562471 homozygote A>G, amino acid pos. 1061, E>E, polymorphism rs7430407
Amplicon 24: pos. 38547178 heterozygote A>G, intron, polymorphism rs41312393
Amplicon 27: pos. 38536074 deletion T, intron
Amplicon 28c: pos. 38592406 T>C, exon, amino acid pos. 1818, D>D, rs1805126
Amplicon 28i: pos. 38530853 deletion C, exon, mRNA pos. 7205, near 3 UTR, nontraslanted region
pos. 38530856 insertion A, exon, mRNA pos. 7202, near 3 UTR, nontraslanted region
pos. 38530974 C>T, exon, mRNA pos. 7084, near 3 UTR, nontraslanted region

9
provocation test
Diagnosed BS
Asymptomatic
provocation test
Patient 7
Patient 8
Major clinical data
Table 2: Continued.
Amplicon 2: pos. 38614716, A>G, amino acid pos. 29, A>A, polymorphism rs6599230
pos. 38614815, G>C, exon, mRNA pos.182, nontraslanted region
Amplicon 24: pos. 38547178 heterozygote A/G, intron, polymorphism rs41312393
Amplicon 28l: pos. 38529996 homozygote C>G, exon, mRNA pos. 8062, near 3 UTR, nontraslanted region
10
11
Table 3: The list of intronic alterations and exchanges in noncoding regions along with short stretches of sequences alignments (WT > MT).
Amplicon
Position
Pos. 38631119
Detected
polymorphism
(WT > MT)
Localization
G>A
Exon,
nontraslanted
region
1
11
Amp. 1 WT CGCAGGCTCAGCGGCC
Amp. 1 MT CGCAGGCTCAACGGCC
cgcaggctca cggcc
Consensus
Exon,
nontraslanted
region
1
11
Amp. 2 WT CCAGAAGCAGGATGAG
Amp. 2 MT CCAGAACCAGGATGAG
ccagaa caggatgag
Consensus
Pos. 38614815
G>C
Pos. 38603806
Pos. 38603801
Insertion A
T>A
Pos. 38604075
G>T
Intron
Intron
Partial alignment (WT > MT)
Amp. 4 WT
Amp. 4 MT
Consensus
Amp. 4 WT
Amp. 4 MT
Consensus
Pos. 38604076
Pos. 38595390
Pos. 38595384
Pos. 38591480
G>T
G>C
G/C
C>G
Intron
Intron
Intron
Pos. 38589643
Insertion A
Intron
Amp. 6 WT
Amp. 6 MT
Pos. 38585647
A>T
Intron
24
27
Pos. 38544050
Pos. 38538672
Pos. 38536074
Pos. 38536077
C>T
A>G
Deletion T
T>A
Intron
Intron
Intron
1
11
CCTCTGACTGTGTGTC
CCTCTCACTGTCTGTC
cctct actgt tgtc
Amp. 7 WT
Amp. 7 MT
1
11
GAACAAGCACGGGGTC
GAACAAGGACGGGGTC
gaacaag acggggtc
Amp. 8 WT
Amp. 8 MT
Amp. 12 WT
Amp. 12 MT
Consensus
22
1
11
AGCACTGGCCTGGCAGT
AGCACTTGCCTGGCAGT
agcact gcctggcagt
Consensus
Consensus
12
ggaaggggg cttg g
1
11
Amp. 4 WT TGGTAGCACTGGCCTGG
Amp. 4 MT TGGTAGCACTGTCCTGG
Consensus tggtagcactg cctgg
Consensus
1
11
GGAAGGGGG-CTTGTG
GGAAGGGGGACTTGAG
1
11
CTGGGTA-TGTGGCA
CTGGGTAATGTGGCA
ctgggta tgtggca
1
11
GCCAGTGGCACAAAAG
GCCAGTGGCTCAAAAG
gccagtggc caaaag
Consensus
1
11
CCATTTCTACTTTG
CCATTTTTACTTTG
ccattt tactttg
Amp. 24 WT
Amp. 24 MT
1
11
GCCAAGCAACCAGG
GCCAAGCAGCCAGG
Consensus
gccaagca ccagg
Amp. 22 WT
Amp. 22 MT
1
11
Amp. 27 WT CCTGCTGAGCACTTTC
Amp. 27 MT CCAGC-GAGCACTTTC
Consensus
cc gc gagcactttc
12

Table 3: Continued.
Amplicon
28f
28g
Position
Pos. 38531693
Pos. 38531355
Detected
polymorphism
(WT > MT)
Localization
A>G
Exon, near
3 UTR,
nontraslanted
region
G>A
Exon, near
3 UTR,
nontraslanted
region
Partial alignment (WT > MT)

Amp. 28f WT
Amp. 28f MT
Consensus
Amp. 28g WT
Amp. 28g MT
Consensus
Amp. 28i WT
Amp. 28i MT
Consensus
28i
28k
28l
Pos. 38530853
Pos. 38530856
Pos. 38530974
Pos. 38531102
Pos. 38530279
Pos. 38529996
Deletion C
insertion A
C>T
C>T
Exon, near
3 UTR,
nontraslanted
region
T>C
3 UTR,
nontraslanted
region
C>G
Exon, near
3 UTR,
nontraslanted
region
the disease symptoms. These mutations/polymorphisms at

introns leading to the disturbances of the splicing process are
described in the disorders of cardiovascular system [19].
In the course of DNA analysis of the SCN5A sodium
gene, the following 6 known polymorphisms were identified:
rs6599230, rs41312393, rs1805126, rs7429945, rs41315485, and
rs7430407. In this group, 3 polymorphisms were observed in
the protein coding regions, 2 in the nontraslanted regions,
and one at the intron. None of them had been associated with
BS before. Also, 8 new genetic variants were found at exons in
the nontraslanted regions, 12 at introns, and 2 in the protein
coding regions that cause no change in the coded amino acid.
None of 3 point mutations (S321Y, S519F, and K974D) in the
protein coding regions that alter the coded amino acid has
been associated previously with BS [5]. According to Zimmer
and Surber, as well as bioinformatic analysis, we are able to
localize their positions in protein sequence. S321Y is localized
in the intracellular loop III, S519F in the intracellular loop IV,
whereas K974D is localized in the C-terminal intracellular
fragment of SCN5a protein. According to the bioinformatic
1
11
GGCCTCAGCCCC
GGCCTCGGCCCC
ggcctc gcccc
1
11
CAAAGCAGAAGTGGAA
CAAAGCAAAAGTGGAA
caaagca aagtggaa
1
11
ATCGGAAGAGAG
AT-GGAAGAGAG
at ggaagagag
1
11
Amp. 28i WT CCCAGCCAGCCAAmp. 28i MT
CCCAGCCAGCCAA
Consensus
cccagccagcca
1
11
Amp. 28i WT
CCTTTTCTTCCCCTCCTG
Amp. 28i MT
CCTTTTTTTCCCCTCCTG
cctttt ttcccctcctg
Consensus
1
11
Amp. 28i WT
GGCCCCCTATTGTCTCCA
Amp. 28i MT
GGCCCCCTATTGTTTCCA
Consensus
ggccccctattgt tcca
Amp. 28k WT
Amp. 28k MT
1
11
TCTCCCATGGAGC
TCTCCCACGGAGC
Consensus
tctccca ggagc
Amp. 28l WT
Amp. 28l MT
1
11
CAGCGACATTTCTC
CAGCGAGATTTCTC
Consensus
cagcga atttctc
results, K974D aa alteration is recognized as highly damaging

for protein function (prediction score amounted to about
1, the highest possible). Two other aa changes were also
reported as possible damage; however, their probability score
amounted from 0.85 to 0.55.
As mentioned previously, also intronic changes could
affect protein function. We performed some basic bioinformatic analyses of detected changes; however, we obtained
contradictory data. Due to the large number of detected
polymorphisms in introns, we decided to perform more
detailed analyses, including in vitro studies.
The majority of detected polymorphisms and genetic
changes found in the study had never been reported as
mutations leading to development of BS. The lack of data
in the literature and the lack of a population control for
this part of the SCN5A gene made it impossible to state
clearly whether the BS syndrome was significantly associated
with the mentioned changes or not. It is also noteworthy
that several genes are associated with BS syndrome; thus,
further genetic study is needed. However, at least new

polymorphisms/mutations that were found in our patients of
a specific phenotype are worth considering.
Special attention ought to be paid to genetic changes
present only in symptomatic patients, for example, with
history of SCA. These genetic changes include the following:
(i) a new polymorphism which involves an alteration in
nucleotides in 38631119 position (G>A) of reference
sequence in 49 mRNA position and developed in a
man with history of SCA and BS diagnosed on the
basis of provocation test result; it was found in none
of the other patients;
(ii) a new genetic variant at exon 28 which involves an
alteration in nucleotides in 38531355 position (G>A)
of reference sequence in 6703 mRNA position and
developed in 5 patients including 2 men with positive
provocation test results and history of SCA as in other
individuals (i.e., the brother and the father of one of
these men);
(iii) two new genetic variants at exon 28c in the protein
coding regions with no alteration in the coded amino
acid (C>T, I1749I; C>T, F1750F) both developed in a
man with history of SCA and positive provocation test
results;
(iv) a new polymorphism at exon 7 which involves an
alteration in nucleotides in 38591480 position (C>G)
of reference sequence and developed in 4 patients out
of 6 individuals with positive ajmaline provocation
test, 3 of these patients had symptomatic BS following
implantation of cardioverter-defibrillator either due
to history of SCA or due to unexplained syncopes;
concurrently the mutation was confirmed in none of
the patients with negative provocation test result.
Considering new data on the role of genetic changes not
only in BS diagnostics but also in prognosis for diagnosed
patients [10, 20], further studies aimed at determining the
role of the identified genetic disorders seem to be extremely
interesting.
5. Conclusions
New genetic variants/polymorphisms in the SCN5A gene
are present in patients with concealed forms of Brugada
Syndrome, yet their role in pathogenesis requires further
studies.
Conflict of Interests
The authors do not report any conflict of interests regarding
the publication of this paper.
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Correspondence should be addressed to Beata Uzibo-Zyczkowska;

Copyright 2014 Beata Uzibo-Zyczkowska

tests with class I medicine are critical in revealing concealed

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resulted in the loss of function of the cardiac sodium channel.

2. Materials and Methods

with positive result of ajmaline challenge test (one patient

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Table 1: Distribution of the examined population depending on

RBBB in ECG (complete

transmembrane regions and secondary structure using three

was ventricular fibrillation. The SCA mechanism in the

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of reference sequence and caused no change in the amino

3.5. New Genetic Variants in Nontraslanted Regions at Exons.

Figure 2: MSSCP separation of exon 2 and exon 7 PCR products.

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was found only in 1 person. Clinically, the person with this

SCN5A gene, presence of 3 unknown genetic variants that

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protein coding region, was observed in 4 patients. The

the coded protein (K974D). BS was diagnosed in 2 of the

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History of sudden cardiac

History of sudden cardiac

Major clinical data

Genetic alterations in DNA sequences of SCN5 amplicons

Table 2: The results of MSSCP genotyping of 41 amplicons representing SCN5A gene.

Major clinical data

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Major clinical data

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Partial alignment (WT > MT)

cctct actgt tgtc

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Partial alignment (WT > MT)

the disease symptoms. These mutations/polymorphisms at

results, K974D aa alteration is recognized as highly damaging

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Submit your manuscripts at

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Research and Treatment

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